Renoprotective effects of laxative linaclotide: Inhibition of acute kidney injury and fibrosis in a rat model of renal ischemia-reperfusion.

Ischemia-reperfusion (I/R) leads to tissue damage in transplanted kidneys, resulting in acute kidney injury (AKI) and chronic graft dysfunction, which critically compromises transplant outcomes, such as graft loss. Linaclotide, a guanylate cyclase C agonist clinically approved as a laxative, has recently been identified to exhibit renoprotective effects in a chronic kidney disease (CKD) model. This study evaluates the therapeutic effects of linaclotide on AKI triggered by I/R in a rat model with an initial comparison with other laxatives. Here, we show that linaclotide administration resulted in substantial reduction in serum creatinine levels, reflective of enhanced renal function. Histological examination revealed diminished tubular damage, and Sirius Red staining confirmed less collagen deposition, collectively indicating preserved structural integrity and mitigation of fibrosis. Further analysis demonstrated lowered expression of TGF-ß and associated fibrotic markers, α-SMA, MMP2, and TIMP1, implicating the downregulation of the fibrogenic TGF-ß pathway by linaclotide. Furthermore, one day after I/R insult, linaclotide profoundly diminished macrophage infiltration and suppressed critical pro-inflammatory cytokines such as TNF, IL-1ß, and IL-6, signifying its potential to disrupt initial inflammatory mechanisms integral to AKI pathology. These findings suggest that linaclotide, with its established safety profile, could extend its benefits beyond gastrointestinal issues and potentially serve as a therapeutic intervention for organ transplantation. Additionally, it could provide immediate and practical insights into selecting laxatives for managing patients with AKI or CKD, regardless of the cause, and for those receiving dialysis or transplant therapy.

Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo.

Extracellular acidification indicates a metabolic shift in cancer cells and is, along with tissue hypoxia, a hallmark of tumor malignancy. Thus, non-invasive mapping of extracellular pH (pHe) is essential for researchers to understand the tumor microenvironment and to monitor tumor response to metabolism-targeting drugs. While electron paramagnetic resonance (EPR) has been successfully used to map pHe in mouse xenograft models, this method is not sensitive enough to map pHe with a moderate amount of exogenous pH-sensitive probes. Here, we show that a modified EPR system achieves twofold higher sensitivity by using the multiple harmonic detection (MHD) method and improves the robustness of pHe mapping in mouse xenograft models. Our results demonstrate that treatment of a mouse xenograft model of human-derived pancreatic ductal adenocarcinoma cells with the carbonic anhydrase IX (CAIX) inhibitor U-104 delays tumor growth with a concurrent tendency toward further extracellular acidification. We anticipate that EPR-based pHe mapping can be expanded to monitor the response of other metabolism-targeting drugs. Furthermore, pHe monitoring can also be used for the development of improved metabolism-targeting cancer treatments.

The efficacy of trimodal chemoradiotherapy with gemcitabine and cisplatin as a bladder-preserving strategy for the treatment of muscle-invasive bladder cancer: a single-arm phase II study.

OBJECTIVE: Radical cystectomy remains the standard treatment for muscle-invasive bladder cancer; however, a substantial number of patients with muscle-invasive bladder cancer are not appropriate candidates to radical cystectomy due to co-morbidities or anxiety regarding bladder preservation. Trimodal bladder-sparing therapy is an intelligent and attractive treatment option for such patients. We established a novel treatment strategy using trimodal treatment with gemcitabine and cisplatin. METHODS: Patients diagnosed with muscle-invasive bladder cancer by transurethral resection of bladder tumor and who wished for bladder preservation were recruited. The regimens were gemcitabine 300 mg/m2 and cisplatin 30 mg/m2 in day 1 and concomitant irradiation 1.8 Gy/Fr, five fractions per week. Irradiation was administered to the true pelvis up to 36 Gy and was then boosted to the entire bladder until a total of 54 Gy. Transurethral resection of bladder tumor was also performed after chemoradiotherapy to evaluate pathological response to treatment. We evaluated treatment efficacy and survival, safety of chemoradiotherapy with gemcitabine and cisplatin. RESULTS: Thirty-eight patients were enrolled, and three patients were excluded. Pathological complete response after chemoradiotherapy was observed in 31 patients, and the 5-year bladder-intact metastasis-free survival rate was 76%. The 5-year cancer-specific and overall survival rates for chemoradiotherapy were 85 and 75%, respectively, which were not significantly different from those for radical cystectomy (73 and 71%, respectively). Grade 3/4 adverse events included neutropenia (63%), anemia (18%) and thrombocytopenia (37%); however, treatment-related deaths were not observed. CONCLUSIONS: Chemoradiotherapy using gemcitabine and cisplatin for muscle-invasive bladder cancer is effective for local cancer control and shows no significant difference in oncological prognosis compared with radical cystectomy.

Hyperpolarized 13 C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in†vivo Study.

The front cover artwork is provided by the group of Dr. Neil J. Stewart, Prof. Hiroshi Hirata, and Dr. Shingo Matsumoto (Hokkaido University, Japan) as well as Dr. Takuya Hashimoto (Chiba University, Japan). The image shows hyperpolarized 13 C fumarate metabolism to hyperpolarized 13 C malate, which is released into the extracellular space in regions of necrotic cell death, where the cell membrane is disrupted. Read the full text of the Article at 10.1002/cphc.202001038.

Hyperpolarized 13 C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in†vivo Study.

Hyperpolarized [1-13 C]fumarate is a promising magnetic resonance imaging (MRI) biomarker for cellular necrosis, which plays an important role in various disease and cancerous pathological processes. To demonstrate the feasibility of MRI of [1-13 C]fumarate metabolism using parahydrogen-induced polarization (PHIP), a low-cost alternative to dissolution dynamic nuclear polarization (dDNP), a cost-effective and high-yield synthetic pathway of hydrogenation precursor [1-13 C]acetylenedicarboxylate (ADC) was developed. The trans-selectivity of the hydrogenation reaction of ADC using a ruthenium-based catalyst was elucidated employing density functional theory (DFT) simulations. A simple PHIP set-up was used to generate hyperpolarized [1-13 C]fumarate at sufficient 13 C polarization for ex vivo detection of hyperpolarized 13 C malate metabolized from fumarate in murine liver tissue homogenates, and in vivo 13 C MR spectroscopy and imaging in a murine model of acetaminophen-induced hepatitis.

Comprehensive analysis of mechanism underlying hypouricemic effect of glucosyl hesperidin.

Hyperuricemia is caused by hepatic overproduction of uric acid and/or underexcretion of urate from the kidneys and small intestine. Although increased intake of citrus fruits, a fructose-rich food, is associated with increased risk of gout in humans, hesperidin, a flavonoid naturally present in citrus fruits, reportedly reduces serum uric acid (SUA) levels by inhibiting xanthine oxidase (XOD) activity in rats. However, the effects of hesperidin on renal and intestinal urate excretion were previously unknown. In this study, we used glucosyl hesperidin (GH), which has greater bioavailability than hesperidin, to clarify comprehensive mechanisms underlying the hypouricemic effects of hesperidin in vivo. GH dose-dependently decreased SUA levels in mice with hyperuricemia induced by potassium oxonate and a fructose-rich diet, and inhibited XOD activity in the liver. GH decreased renal urate excretion without changes in kidney URAT1, ABCG2 or GLUT9 expressions, suggesting that reducing uric acid pool size by inhibiting XOD decreased renal urate excretion. We also found that GH had no effect on intestinal urate excretion or protein expression of ABCG2. Therefore, we concluded that GH exhibits a hypouricemic effect by inhibiting XOD activity in the liver without increasing renal or intestinal urate excretion. Of note, this is the first study to elucidate the effect of a flavonoid on intestinal urate excretion using a mice model, whose findings should prove useful in future food science research in the area of urate metabolism. Taking these findings together, GH may be useful for preventing hyperuricemia, especially in people with the overproduction type.

In vitro simultaneous mapping of the partial pressure of oxygen, pH and inorganic phosphate using electron paramagnetic resonance.

The partial pressure of oxygen (pO2) and the extracellular pH in the tumour microenvironment are essential parameters for understanding the physiological state of a solid tumour. Also, phosphate-containing metabolites are involved in energy metabolism, and interstitial inorganic phosphate (Pi) is an informative marker for tumour growth. This article describes the simultaneous mapping of pO2, pH and Pi using 750 MHz continuous-wave (CW) electron paramagnetic resonance (EPR) and a multifunctional probe, monophosphonated trityl radical p1TAM-D. The concept was demonstrated by acquiring three-dimensional (3D) maps of pO2, pH and Pi for multiple solution samples. This was made possible by combining a multifunctional radical probe, fast CW-EPR spectral acquisition, four-dimensional (4D) spectral-spatial image reconstruction, and spectral fitting. The experimental results of mapping pO2, pH and Pi suggest that the concept of simultaneous mapping using EPR is potentially applicable for the multifunctional measurements of a mouse tumour model.

In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance.

An electron paramagnetic resonance (EPR)-based method for noninvasive three-dimensional extracellular pH mapping was developed using a pH-sensitive nitroxyl radical as an exogenous paramagnetic probe. Fast projection scanning with a constant magnetic field sweep enabled the acquisition of four-dimensional (3D spatial +1D spectral) EPR images within 7.5 min. Three-dimensional maps of pH were reconstructed by processing the pH-dependent spectral information on the images. To demonstrate the proposed method of pH mapping, the progress of extracellular acidosis in tumor-bearing mouse legs was studied. Furthermore, extracellular pH mapping was used to visualize the spatial distribution of acidification in different tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells. The proposed EPR-based pH mapping method enabled quantitative visualization of regional changes in extracellular pH associated with altered tumor metabolism.

Impact of divergent differentiation in urothelial carcinoma on oncological outcome in patients with T1 high-grade bladder cancer.

OBJECTIVE: T1 high-grade bladder cancer has a poor prognosis compared with other non-muscle-invasive bladder cancers. We investigated the clinical outcomes among patients with T1 high-grade bladder cancer to identify factors related to cancer recurrence and disease progression. METHODS: We retrospectively reviewed the data of 148 patients who were diagnosed with T1 high-grade bladder cancer by transurethral resection from January 2001 to February 2015 at our institution. Clinicopathological factors were analyzed using univariate and multivariate analyses. RESULTS: The median age and follow-up duration were 72 years and 45.4 months, respectively. Sixty-two patients (41.9%) had recurrence, 28 (18.9%) experienced progression and 13 (8.8%) died of bladder cancer. In the multivariate analysis, divergent differentiation was an independent factor related to recurrence (P = 0.0096, hazard ratio = 2.5), whereas a non-papillary tumor shape, divergent differentiation and presence of a residual tumor at the time of the second transurethral resection were independent factors related to progression (P = 0.0349, hazard ratio = 3.8; P = 0.0074, hazard ratio = 6.8 and P = 0.0449, hazard ratio = 4.1, respectively). There were no independent factors related to cancer-specific mortality. Divergent differentiation was the only independent factor associated with both recurrence and progression. In addition, patients with divergent differentiation had significantly worse recurrence-free survival and progression-free survival rates than did patients without divergent differentiation (log-rank P = 0.0009 and P = 0.0019, respectively). CONCLUSIONS: In this study, the presence of divergent differentiation was related to worse oncological outcomes in patients with T1 high-grade bladder cancer. Patients with divergent differentiation may require stringent follow-up and early cystectomy after recurrence to improve oncological outcomes.

Feasibility of in vivo three-dimensional T 2* mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging.

OBJECTIVES: The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T 2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. MATERIALS AND METHODS: Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-(1-15N)-1-oxyl (2H,15N-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-1-oxyl (2H-DCP), were used in the study. A clonogenic cell survival assay was performed with the 2H-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected 2H,15N-DCP and 2H-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T 2* mapping. RESULTS: 2H-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of 2H,15N-DCP in tumor tissues was 24.7 ± 2.9 min (mean ± standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the 2H,15N-DCP radical showed a plateau of 10.2 ± 1.2 min (mean ± SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T 2* maps with 2H,15N-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. CONCLUSION: EPR signals of 2H,15N-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T 2* mapping with CW-EPR-based SPI.

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961.

Novel blood-brain barrier-permeable spin probe for in vivo electron paramagnetic resonance imaging.

A novel blood-brain barrier (BBB)-permeable compound 10 was discovered, wherein the nitroxide moiety was linked to a nicotine acetylcholine receptor ligand. It was applied as a probe for electron paramagnetic resonance (EPR) imaging of the mouse brain. The results demonstrated that the newly synthesized compound 10 exhibited BBB permeability. These findings provide an essential discovery for in vivo EPR imaging.

BUBR1 overexpression predicts disease-specific survival after nephroureterectomy in patients with upper tract urothelial carcinoma.

OBJECTIVE: To date, there are few reliable markers to distinguish tumors with aggressive characteristics in upper tract urothelial carcinoma. The purpose of this study was to identify a biomarker related to genetic instability (chromosomal instability or microsatellite instability) with prognostic value, in patients with upper tract urothelial carcinoma. METHODS: Expression of chromosomal instability-related markers (BUBR1, p53, polo-like kinase 1) and microsatellite instability-related markers (mismatch repair proteins, MLH1 and MSH2) were assessed by immunohistochemistry in 100 patients who had radical nephroureterectomy for upper tract urothelial carcinoma. Numerical aberrations of chromosomes 7, 9 and 17 were evaluated by fluorescence in situ hybridization, which allowed an estimation of the degree of chromosomal instability. BUB1B copy number was examined by array-based comparative genomic hybridization in 32 patients with upper tract urothelial carcinoma. RESULTS: BUBR1 status was most significantly correlated with chromosomal instability-related and low mismatch repair parameters, according to the molecular biomarkers examined. Overexpression of BUBR1 is frequently detected in tumors with higher histological grade (P < 0.0001) and is significantly associated with chromosomal instability (P = 0.0071). Array-based comparative genomic hybridization revealed that no tumors (0%) showed BUB1B amplification and gain, indicating that overexpression of BUBR1 was independent of BUB1B copy number. For disease-specific survival, BUBR1 overexpression, lymphovascular invasion, pathological tumor stage, pathological lymph node involvement and low MSH2 expression were significant prognostic factors in univariate analyses. In multivariate analyses, BUBR1 overexpression was an independent prognostic factor for disease-specific survival (P = 0.0483, risk ratio 3.76, 95% confidence interval: 1.01-18.43). CONCLUSIONS: BUBR1 may have significant potential as a biomarker for estimating disease-specific survival in patients with upper tract urothelial carcinoma treated by radical nephroureterectomy.

Biosynthesis of floral scent 2-phenylethanol in rose flowers.

Plants emit chemically diverse volatile compounds for attracting pollinators or putting up a chemical defense against herbivores. 2-Phenylethanol (2PE) is one of the abundantly emitted scent compounds in rose flowers. Feeding experiments with l-[(2)H8]phenylalanine into rose flowers and subsequent analysis using gas chromatography-mass spectrometry analysis revealed the hypothetical biosynthetic intermediates to [(2)H8]-2PE, and the biochemical and genetic analyses elucidated the principal pathway to [(2)H8]-2PE. We recently found season-specific 2PE pathway producing [(2)H7]-2PE from l-[(2)H8]phenylalanine. This is a unique example where the dominant pathway to a specific compound changes with the seasons. This review focuses on the biosynthesis of floral volatiles and their regulation to adapt to the changes in the environment.

CYP1B1 promotes tumorigenesis via altered expression of CDC20 and DAPK1 genes in renal cell carcinoma.

BACKGROUND: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. METHODS: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses. RESULTS: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples. CONCLUSIONS: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.

Four-channel surface coil array for 300-MHz pulsed EPR imaging: proof-of-concept experiments.

Time-domain electron paramagnetic resonance imaging is currently a useful preclinical molecular imaging modality in experimental animals such as mice and is capable of quantitatively mapping hypoxia in tumor implants. The microseconds range relaxation times (T1 and T2) of paramagnetic tracers and the large bandwidths (tens of MHz) to be excited by electron paramagnetic resonance pulses for spatial encoding makes imaging of large objects a challenging task. The possibility of using multiple array coils to permit studies on large sized object is the purpose of the present work. Toward this end, the use of planar array coils in different configurations to image larger objects than cannot be fully covered by a single resonator element is explored. Multiple circular surface coils, which are arranged in a plane or at suitable angles mimicking a volume resonator, are used in imaging a phantom and a tumor-bearing mouse leg. The image was formed by combining the images collected from the individual coils with suitable scaling. The results support such a possibility. By multiplexing or interleaving the measurements from each element of such array resonators, one can scale up the size of the subject and at the same time reduce the radiofrequency power requirements and increase the sensitivity.

In vivo tumour extracellular pH monitoring using electron paramagnetic resonance: the effect of X-ray irradiation.

The in vivo quantification of extracellular pH (pHe ) in tumours may provide a useful biomarker for tumour cell metabolism. In this study, we assessed the viability of continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy with a pH-sensitive nitroxide for the measurement of extracellular tumour pH in a mouse model. CW-EPR spectroscopy (750 MHz) of C3H HeJ mice hind leg squamous cell tumour was performed after intravenous tail vein injection of pH-sensitive nitroxide (R-SG, 2-(4-((2-(4-amino-4-carboxybutanamido)-3-(carboxymethylamino)-3-oxoproylthio)methyl)phenyl)-4-pyrrolidino-2,5,5-triethyl-2,5-dihydro-1Н-imidazol-1-oxyl) during stages of normal tumour growth and in response to a single 10-Gy dose of X-ray irradiation. An inverse relationship was observed between tumour volume and pHe value, whereby, during normal tumour growth, a constant reduction in pHe was observed. This relationship was disrupted by X-ray irradiation and, from 2-3 days post-exposure, a transitory increase in pHe was observed. In this study, we demonstrated the viability of CW-EPR spectroscopy using R-SG nitroxide to obtain high-sensitivity pH measurements in a mouse tumour model with an accuracy of <0.1 pH units. In addition, the measured changes in pHe in response to X-ray irradiation suggest that this may offer a useful method for the assessment of the physiological change in response to existing and novel cancer therapies.

Cytochrome P450 1B1 polymorphisms and risk of renal cell carcinoma in men.

The cytochrome P450 1B1 (CYP1B1) enzyme activates xenobiotics to reactive forms as well as convert estradiol to 4-hydroxy-estradiol that has been shown to play a role in the carcinogenesis process of the kidney in male but not female animals. Prior reports show polymorphic variants of CYP1B1 to alter catalytic activity, and thus, we hypothesize that polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell in men. The genetic distributions of five CYP1B1 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 480 normal healthy subjects and 403 sporadic renal cell carcinoma cases. All subjects were Caucasian men. The sites evaluated were codons 48 (C → G, Arg → Gly, rs10012), 119 (G → T, Ala → Ser, rs1056827), 432 (C → G, Leu → Val, rs1056836), 449 (C → T, Asp, rs1056837), and 453 (A → G, Asn → Ser, rs1800440). A trend was demonstrated for the 432 Val/Val (χ2, P = 0.06) and 449 T/T (χ2, P = 0.1) genotypes to play a protective role against renal cancer. Odds ratio (95 % confidence interval) for Val/Val compared to Leu/Leu at codon 432 was 0.65 (0.44-0.95) and T/T compared to C/C at codon 449 was 0.67 (0.45-0.99). Codons 432 and 449 were observed to be linked (D = 0.24), and haplotype involving 432 Val and 449 T was significantly reduced in cancer cases (P = 0.04). No association was found, however, when analyzing polymorphic sites with clinical stage of cancer. These results demonstrate polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of renal cell carcinoma.

Proton-Electron Double-Resonance Imaging of pH using phosphonated trityl probe.

Variable Radio Frequency Proton-Electron Double-Resonance Imaging (VRF PEDRI) enables extracting a functional map from a limited number of images acquired at pre-selected EPR frequencies using specifically designed paramagnetic probes with high quality spatial resolution and short acquisition times. In this work we explored potential of VRF PEDRI for pH mapping of aqueous samples using recently synthesized pH-sensitive phosphonated trityl radical, pTR. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of pTR probe allows for a pH map extraction. Long relaxation times of pTR allow for pH mapping at EPR irradiation power as low as 1.25 W during 130 s acquisition time with spatial resolution of about 1 mm. This is particularly important for in vivo applications enabling one to avoid sample overheating by reducing RF power deposition.